The role of beta arrestin 1 in vascular tone regulation of pulmonary arteries

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The role of beta arrestin 1 in vascular tone regulation of pulmonary arteries

Dissertation zur

Erlangung des Doktorgrades (Dr. rer. nat.) der

Mathematisch-Naturwissenschaftlichen Fakultät der

Rheinischen Friederich-Wilhelms-Universität Bonn

vorgelegt von

Leonard Lebender

aus

Bonn, Deutschland

Bonn 2021

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Angefertigt mit der Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn

1. Gutachter: Prof. Dr. Bernd K. Fleischmann 2. Gutachterin: Prof. Dr. Evi Kostenis

Tag der Promotion: 30.08.2021 Erscheinungsjahr: 2021

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Acknowledgements

I

Acknowledgements

Mein erster Dank gilt Herrn Prof. Fleischmann für seine Unterstützung als Doktorvater und als erster Gutachter dieser Arbeit, sowie Frau Prof. Daniela Wenzel für die enge und hervorragende Betreuung und stetige Unterstützung dieses Projektes. Ich danke weiterhin Frau Prof. Evi Kostenis für Ihre Betreuung als Co-supervisor und die tolle Zusammenarbeit. Dank gilt auch der fachlichen und finanziellen Unterstützung durch die DFG und das Graduiertenkolleg 1873 unter der Leitung von Prof.

Alexander Pfeifer, welches erst diese Arbeit ermöglicht hat. Besonderen Dank möchte ich auch Herrn Prof. Volkmar Gieselmann aussprechen, welcher mir stets bei vielen Fragen zur Seite stand. Durch die enge Zusammenarbeit mit der Biochemie wurde dieses Projekt erst zu dem, was es heute ist.

Den Mitpromovierenden des GRK1873 möchte ich für die tolle gemeinsame Zeit und die moralische Unterstützung danken, wodurch bei unzähligen Seminaren, Vorträgen und Ausflügen nach Feierabend viel Freude aufkam.

Meine Arbeit als Doktorand wurde von der gemeinsamen Zeit mit den lieben Kollegen, welche mich stets begleiteten, sehr bereichert. Ich hatte großes Glück, in ein so tolles Team aufgenommen zu werden und habe viele schöne gemeinsame Stunden verbracht- sei es bei Franks Dinnerpartys, Elas DJ-Sets, Yokes Grillpartys oder der gemeinsamen Weihnachtsfeier. Vielen Dank Euch allen für diese aufregende Zeit!

Besonders hervorheben möchte ich die Arbeitsgruppe Wenzel, namentlich Jennifer Dietrich, Alexander Seidinger, Annika Simon, Dr. Sarah Rieck, Dr. Ela Matthey und Prof. Daniela Wenzel. Ich danke für die unschätzbare Unterstützung und den Zusammenhalt während meiner Doktorandenzeit. Gemeinsame Ausflüge, Konferenzen, aber auch die studentische Lehre und der Laboralltag waren ein tolles Erlebnis mit Euch!

Zum Schluss möchte ich meinen Freunden, meiner Familie sowie der Familie Prünte für die stetige Motivation, das offene Ohr und die emotionale Unterstützung danken. Eurem Rückhalt verdanke ich die Vollendung dieser Arbeit, Ihr seid großartig!

Letzter und größter Dank geht an Laura, welche mich in allen Situationen stets begleitet und ermutigt hat diesen Weg zu gehen! Du hast die gemeinsame Doktorzeit zu etwas Besonderem werden lassen, Du bist wunderbar!

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Table of contents

II

Abbreviations

°C Degrees Celsius µL Microliter µm Micrometer µM Micromolar AC Adenylyl cyclase ACh Acetyl choline ANOVA Analysis of variance AP Alkaline phosphates apo-sGC heme-deficient sGC APS Ammonium persulfate

AT1A Angiotensin II receptor type 1A beta arr1 Beta arrestin 1

beta arr2 Beta arrestin 2 bmp Beats per minute BSA Bovine serum albumin ca. Circa

CaCl2 Calcium chloride CaM Calmodulin

cAMP Cyclic adenosine monophosphate cDNA Complementary desoxyribunucleic

acid

cGMP Cyclic guanosine monophosphate CMC Carboxymethyl cellulose

CTEPH Chronic thromboembolic pulmonary hypertension

Cyb5r3 Cytochrome b5 reductase 3 DAG Diacylglycerol

dH2O Aqua destillata (distilled water) DMEM Dulbecco's Modified Eagle Medium DMSO Dimethyl sulfoxide

DNA Desoxyribunucleic acid

DPBS Dulbecco’s Phosphate Buffered Saline

EDTA Ethylenediaminetetraacetic acid ELISA Enzyme linked immunosorbent

assay

et al. And others ("et alii") EtOH Ethanol

FACS Fluorescense-activated cell sorting FCS Fetal calf serum

Fsk Forskoline

g Gravity

GAP GTPase activating protein GDP Guanosine diphosphate

GEF Guanine-nucleotide exchange factor

GFP Green fluorescent protein GIRK G-protein coupled inwardly

rectifying potassium channel GPCR G protein-coupled receptor

G protein Guanine nucleotide binding protein GRK G protein-coupled receptor kinases GTP Guanosine triphosphate

h Hour

H&E Hematoxylin and eosin H2O2 Hydrogen peroxide HCl Hydrochloric acid

HEK293 Human embryonic kidney 293 cells

Hx Hypoxia

i.p. Intraperitoneal i.v. Intravenous

IB Immunoblot

IBMX 3-isobutyl-1-methyxanthine InsP3R Inositol triphosphate receptor IP Prostacycline receptor

IP3 Inositol trisphosphate

IRAG InsP3R- associated PKG substrate isoform A

JNK c-Jun N-terminal kinase KCl Potassium chloride kg Kilogram

KO Knockout

LB Lysogeny broth LV Left ventricle

LVSP Left ventricular systolic pressure MAPK Mitogen-activated protein kinase MBP Maltose Binding Protein

MCh Methacholine MgCl2 Magnesium chloride MgSO4 Magnesium sulfate

min Minute

MLC20 Regulatory myosin light chain MLCP Myosin light chain phosphatase mM Millimolar

mmHg Millimeter mercury mN Millinewton

mPASMC Murine pulmonary artery smooth muscle cell mRNA Messenger RNA

msec Milisecond

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Abbreviations

VII Na2HPO4 Sodium hydrogen phosphate

NaCl Sodium chloride NaHCO3 Sodium bicarbonate NaIO3 Sodium iodate NaOH Sodium hydroxide

nM Nanomolar

Nx Normoxia

O2 Oxygen

p Passage

PA Pulmonary artery

PAGE Polyacrylamide gel electrophoresis PAH Pulmonary arterial hypertension PAPm Resting mean pulmonary arterial

pressure

PBS Phosphate-buffered saline PCR Polymerase chain reaction PDE Phosphodiesterase PDE4D Phosphodiesterase 4D

PDGF Platelet derived growth factor Pen-Strep Penicillin-Streptomycin PFA Paraformaldehyde PH Pulmonary hypertension PIP2 Phospohatidylinositol-4,5-

bisphosphate PKA Protein kinase A PKC Protein kinase C PKG Protein kinase G PLB Phospholamban PLC Phospholipase C PLCβ Phospholipase Cβ pNpp P-nitrophenyl phosphate PSS Physiological salt solution PSS low

Ca2+

Physiological salt solution low Calcium

PTFE Polytetrafluorethylen PVDF-FL Polyvinylidene fluoride low fluorescence PVR Pulmonary vascular resistance qPCR Quantitative polymerase chain

reaction

RhoA Ras homolog family member A

RIPA Radioimmunoprecipitation assay buffer

ROCK Rho-kinase Roflu Roflumilast

rpm Rounds per minute RT Room temperature

RT qPCR Real-time quantitative polymerase chain reaction RV Right ventricle

RVSP Right ventricular systolic pressure s.c. Subcutan

s.e.m. Standard error of the mean

S.O.C. Super optimal broth with catabolite repression

SDS Sodium dodecyl sulfate

sec Second

SERCA Sarco(endo)plasmic reticulum Ca²⁺- ATPase

sGC alpha1 Soluble guanylyl cyclase subunit alpha1

sGC alpha1 beta 1

Soluble guanylyl cyclase alpha1 beta1

sGC alpha2 Soluble guanylyl cyclase subunit alpha2

sGC beta1 Soluble guanylyl cyclase subunit beta1

SNP Sodium nitroprusside SPF Specific-pathogen-free TBS Tris-buffered saline

TBST Tris-buffered saline + 0.1% tween- 20

TEMED N,N,N',N'-Tetramethyl ethylenediamine

Tris Tris(hydroxymethyl)aminomethane U International units

V Volt

VEGFR3 vascular endothelial growth factor receptor 3 VSMC Vascular smooth muscle cell WB Western blot

Δbeta

Arr1/2 Beta arrestin 1 and beta arrestin 2 double knockout

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Introduction

1

1 Introduction

1.1 Physiology of the vascular system

The vascular system consists of extensive networks with proper blood flow serving to maintain cellular homeostasis in the body .¹ Larger vessels mainly enable blood transport whereas smaller capillaries facilitate substance and nutrient exchange.² The vascular system of the body consists of a systemic circuit that enables blood supply to all tissues, and a pulmonary circuit that enables blood gas exchange in the lung.³

1.1.1 The systemic circuit

The heart pumps blood from the left ventricle through arteries, capillaries and veins in a circuit back to the right atrium.² Blood vessels highly differ in their structure depending on their function in the blood circuit. Large arteries are characterized by a high mean blood pressure (100 mmHg) and are subclassified as elastic and muscular types. Elastic arteries are responsible for the transport of large blood volumes and their high elasticity further enables continuous blood transport by dampening the large oscillations during systole and diastole. Muscular arteries are mainly responsible for rapid blood distribution. Compliance describes the adaptation of the vessel volume in response to blood pressure changes.¹ Thus, elastic arteries have a greater compliance compared to muscular arteries. Arteries split into smaller arterioles which again branch into capillaries. The blood pressure is highest in arteries and declines after the small resistance arteries. This is necessary for protection of the thin-walled capillaries, which connect the arterial with the venous system and enable solute and water exchange.^1,2 Capillaries and veins are both characterized by a low mean blood pressure (25 to 2 mm Hg). Leaving the capillaries, the blood returns to the heart through the venous system. Veins act as a reservoir for blood and prevent extensive pooling of blood in the extremities, some contain uni-directional valves.²

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Introduction

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Figure 1: Cross section of an artery.

Arteries are typically divided in three different layers named tunica intima, tunica media and tunica adventitia. Each layer consists of different cell types and a complex interplay of intercellular signaling enables proper functionality regarding tone regulation, cellular homeostasis and substance exchange.

The structure of larger arteries is composed of three layers (Figure 1): The tunica intima mainly consists of endothelial cells, stratum subendotheliale and membrana elastica. Its main purposes are providing a primary diffusion barrier, regulation of blood coagulation, secretion of vasoreactive substances and participation in angiogenesis.^1,4 The tunica media mainly consists of smooth muscle cells and extracellular matrix and is essentially responsible for regulation of the vessel diameter by contraction or relaxation of muscle fibres. The Tunica adventitia contains connective tissue and fibroblasts and has supporting and supply function.^1,4 Capillaries are characterized by a lack of smooth muscle cells, consisting only of a single layer of endothelial cells and a basement membrane. The large total surface area and the rather weak barrier function enable the characteristic exchange of water and substances by diffusion.¹ Veins are structured, similar to arteries, in three different layers, but contain less smooth muscle and connective tissue. Thus, the vascular wall of veins is thinner than arterial walls resulting in a lower median blood pressure in the venous system.¹

1.1.2 The pulmonary circuit

Deoxygenated blood is pumped from the right ventricle through pulmonary arteries (PAs) in a circuit to the left atrium. On its way, blood moves from large PAs to pulmonary capillaries, where an efficient gas exchange takes place in the alveoli. Reoxygenated blood is transported to the left atrium through pulmonary veins and is then distributed from the left ventricle to the systemic blood circuit.² The rather thin-walled and elastic pulmonary vessels are characterized by a high compliance and low resistance.⁵ Due to the low resistance of pulmonary vessels, the mean blood pressure in PAs is much lower (15 mm Hg) than in their systemic counterpart.² The blood supply for organs like liver or brain is regulated by myogenic autoregulation, this mechanism enables a constant blood supply independent of blood pressure changes. This is different in the pulmonary system: blood pressure is kept relatively constant, but to meet an increased demand of oxygenated blood, vessels react passively to given pressure by

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Introduction

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distention. An increase in cardiac output results in increased vessel diameter, while the pressure remains constant.⁶ The main purpose of the pulmonary circuit is the exchange of oxygen and carbon dioxide; to optimize this process, perfusion and ventilation of the lungs have to be adjusted. While the ventilation of individual lung areas and thus the oxygen supply varies, the pulmonary circuit displays the unique phenomenon of hypoxic vasoconstriction (also known as Euler-Liljestrand mechanism): This mechanism ensures optimal oxygenation of the blood by adaption of perfusion. Vessels in areas with low partial pressure of oxygen (pO2) constrict to redirect the blood to lung areas with higher oxygen supply, which improves gas exchange.

While systemic arteries and PAs share the basic three-layered wall structure, PAs have lower and more constant mean blood pressure, higher compliance and lower resistance than systemic arteries. In total, PAs are highly fragile yet very elastic vessels with previously introduced unique properties to enable optimal blood supply for the oxygenation process.

1.1.3 Molecular mechanisms of vascular tone regulation

Control of vascular tone is a complex process modulated by various endogenous compounds. Vascular tone is highly dynamic and adapts to the current physiological state. A complex interplay between constriction and relaxation of different vessels enables precise adaptation to altered demand of blood supply upon various physiological circumstances.¹

Modulation of the blood circuit in general is achieved by local, hormonal or neuronal signals. Local signals such as release of nitric oxide (NO) and adenosine or changes in pO2 are responsible for metabolic adaptation to local physiological changes. Hormones affect vessel tone either directly by receptor activation on the SMC surface such as adrenaline binding to beta2-or alpha1-receptors or indirectly by hormone-dependent release of local vasoactive substances such as endothelial endothelin-1-release caused by increased concentrations of antidiuretic hormone.^2,7 Neuronal control is mainly provided by sympathetic innervation and neuronal feedback mechanisms.²

1.1.3.1 Cross-bridge mechanism of smooth muscle cell contraction and relaxation

Tone regulation of vessels is mainly driven by contraction or dilatation of smooth muscle cell layers in the tunica media. The mechanism of cross-bridge cycling describes the attachment of myosin filament heads (also called cross-bridges) to actin filaments and the sliding of the actin filaments result in the development of force.^8,9 Cross-bridge cycling enables adjustment of the vessel diameter.¹⁰ Muscle contraction is initiated by rising Ca²⁺ concentrations from intracellular stores or via channel transport from the extracellular space. Ca²⁺ ions interact with calmodulin and this complex activates myosin light chain kinase (MLCK). MLCK phosphorylates the regulatory light chain of myosin (MLC20) and thus

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Introduction

4

enables the myosin-actin interaction causing muscle contraction.^2,10 Relaxation of the smooth muscle is driven by a decrease of the intracellular Ca²⁺ concentration and an increase of myosin light chain phosphatase (MLCP) activity. MLCP dephosphorylates the MLC20 and thereby prevents further myosin- actin interaction. Prolongation of contraction is further supported by inhibition of MLCP via Rho kinase activity.¹⁰

Intracellular Ca²⁺ concentrations are directly and indirectly regulated by either activation or inhibition of different receptors on the cell surfaces of vascular smooth muscle and endothelial cells.

1.2 G protein-coupled receptors (GPCRs) and their role in vascular smooth muscle cell (VSMC) tone regulation

Vascular tone is determined by a balance between vasoconstriction and vasorelaxation, which is mediated via various vasoactive substances. Most of them act through GPCRs activating different downstream signaling pathways. Therefore, GPCRs and their modulation play a crucial role in tone regulation of VSMCs.

1.2.1 GPCR signal transduction

GPCRs are the largest, diverse class of transmembrane receptors and play an essential role in physiology. As more than 25% of the FDA approved drugs target GPCRs, they are crucial molecules for today’s treatment of diseases.¹¹

GPCRs consist of seven conserved transmembrane protein helices located within the cellular membrane. Upon ligand binding at the extracellular site of the receptor, a signal is transduced to the intracellular site via conformational change of the receptor. Further signal transduction of the activated GPCR is mediated by intracellular dissociation of heterotrimeric guanine-nucleotide binding proteins (G proteins). ^12,13

Heterotrimeric G proteins consist of different subunits: The Gα subunit contains a high affinity binding site for guanosine nucleotides and binds guanosine diphosphate (GDP) in its inactive form. The Gβγ subunit, consisting of Gβ and Gγ, forms a tight complex and is regarded to be one unit. All three subunits are bound together and form the inactive G protein.¹⁴ G proteins are divided in four major subclasses (Gαs, Gαi/o, Gαq/11 and Gα12/13) on base of sequence similiarity and downstream targets.12,13,15,16

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Introduction

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Figure 2: Process of GPCR activation.

GPCRs are located within the cellular membrane. In their inactive state, GPCRs are separated from their signaling effectors, the G proteins. Activation of the GPCR causes a conformational change of the receptor, resulting in binding of the G protein and exchange of GDP for GTP at the Gα-subunit. The GTP-bound and activated Gα-subunit dissociates from the G protein complex and both subunits, Gα and Gβγ, cause GPCR-initiated downstream signaling. Time-dependent cleavage of GTP to GDP and phosphate at the Gα-subunit terminates the signaling process and both G protein subunits reassemble.

Activation of the GPCR by a ligand enables binding of trimeric G proteins to the receptor.¹⁷ A conformational change of the GPCR results also in rearrangement of transmembrane helices on the cytoplasmic site of the receptor. Especially the outward shift of transmembrane helix 6, along with other helix rearrangements, result in the exposure of an intracellular binding pocket that attracts G proteins, G protein coupled receptor kinases (GRKs) and beta arrestins.¹⁶ While G proteins are the prime signaling effectors of activated GPCRs, GRKs and beta arrestins regulate GPCR activity.

The activated GPCR functions as a guanine-nucleotide exchange factor (GEF), causing an exchange of the bound GDP at the Gα subunit for guanosine triphosphate (GTP).¹⁸ Next, the G protein complex dissociates and both resulting subunits, the GTP-binding Gα- and the Gβγ-subunit, induce further downstream signaling by activating or blocking various proteins. Both activated G protein subunits (Gα and Gβγ) have distinct signaling functions: Gα-subunits regulate andenylyl cyclases (AC),

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Introduction

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phospholipase Cβ (PLCβ), cGMP phosphodiesterases or RhoGEFs. The Gβγ-subunit recruits GRKs, modulates G protein-coupled inwardly rectifying potassium (GIRK) channels and voltage-dependent Ca²⁺channels, regulates ACs, phospholipases, phosphoinositide 3 kinase and mitogen-activated protein kinases (MAPK), to name a few. ¹⁶ The G protein-unbound and still active receptor can bind and activate another G protein, providing an amplification of the signal.¹⁹

These signaling processes of the G protein subunits are terminated by a time-dependent exchange of GTP to GDP at the Gα subunit. The GTP-bound Gα-subunit has a GTPase activity and is further catalyzed by GTPase activating proteins (GAP proteins), resulting in hydrolysis of the bound GTP and thus terminating the signaling process. Upon cleavage of the bound GTP, the inactive complex of G proteins associates (Figure 2). ^12–15

A signal turn-off of activated GPCRs is achieved by rapid phosphorylation of intracellular seryl-threonyl sites of active GPCRs by GRKs and subsequent beta arrestin binding.^19,20

1.2.2 Regulators of GPCR activity: GRKs and beta arrestins

GRKs belong to a family of seven homologs and while GRK isoform 1 and 7 phosphorylate visual opsins, GRK isoform 2-6 target GPCRs and other cell-surface receptors. Arrestins belong to a family of four homologous arrestin proteins. The two visual arrestins (arrestin 1 and 4) target primarily photoreceptors whereas the two non-visual arrestins (arrestin 2 and 3; synonyms beta Arr1 and 2) interact primarily with GPCRs. GRKs and beta arrestins serve as universal GPCR regulators.

Arrestins were first discovered in the 1980s by Kühn et al. as proteins that are actively involved in the turn-off of light-activated rhodopsin in the retina²¹. Pioneer work on the beta2-adrenergic receptor discovered arrestin function aside from inactivation of retinal rhodopsin and allowed for the classification of visual arrestins and non-visual (beta) arrestins, which is still valid today. Cell culture experiments in the early 1990s by the nobel laureates B. K. Kobilka and R. J. Lefkowitz among others led to the discovery of today’s best-known beta arrestin functions: GPCR desensitization and internalization.^22,23 The discovery of beta arrestin function is closely connected with the history of GRKs. It was nearly simultaneously discovered that beta arrestins and GRKs are modulators of activated GPCRs and are required for GPCR uncoupling activity.^24,25 Around 1999, first papers were published suggesting a role for beta arrestins as scaffolding molecules, modulating different downstream signaling pathways.²⁶

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Introduction

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1.2.2.1 Beta arrestin and GRK-facilitated GPCR desensitization

Figure 3: Versatile functions of beta arrestins.

Beta arrestins facilitate receptor desensitization and internalization and act as scaffolds for downstream signaling pathways. (A) Receptor desensitization is driven by beta arrestin-binding to the activated and phosphorylated GPCR. The bound beta arrestin blocks the G protein binding site and prevents further downstream signaling. (B) GPCR internalization is facilitated by beta arrestin-binding and thus transport of the receptor from the cell surface to the cytoplasm in clathrin- coated vesicles. (C) Recent literature suggests scaffolding functions of beta arrestins by i.e. interaction with mitogen- activated protein kinases (MAPPK) or recruitment of phosphodiesterase 4D (PDE4D).

GPCRs undergo a conformational change upon agonist-binding resulting in the dissociation of the G protein into subunits leading to various downstream signaling cascades. Activated GPCRs are under control of GRKs that are recruited by Gβγ subunits to the active receptor and phosphorylate multiple sites in the C-terminus and intracellular loops of the GPCR. This is followed by beta arrestin recruitment and binding to the GPCR, blocking further G protein binding by steric obstruction of the binding site.

This process is known as receptor desensitization, as further G protein-mediated downstream signaling is inhibited by beta arrestin binding to the receptor (Figure 3A).^20,27 While different phosphorylation patterns are suggested to affect fine-tuning of beta arrestin binding, a recent study with rhodopsin suggests two key phosphorylation sites (pT340 and pS343) at the C-terminus necessary for beta arrestin binding.²⁸ Typical beta arrestin recruitment is a rather fast-paced mechanism and occurs within minutes.^29,30

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Introduction

8 1.2.2.2 Beta arrestin-facilitated GPCR internalization

Besides receptor desensitization, beta arrestin binding also facilitates endocytosis of activated GPCRs.

Indeed, beta arrestins are essential for receptor internalization upon agonist-induced receptor activation.³¹ Beta arrestin binding to the GPCR enables beta arrestin interaction with adaptor protein 2 (AP2) which promotes endocytosis of the receptor-complex (Figure 3B). AP2 is a clathrin-adaptor that facilitates formation of clathrin-coated pits at the cell membrane, causing invagination of the activated GPCR. This is followed either by receptor recycling and new recruitment to the cell membrane or receptor degradation. Two patterns of receptor recruitment are described: Class A receptors (i.e. beta2-adrenergic receptors) are known for their low-affinity beta arrestin binding resulting in rather rapid recycling of the receptor upon internalization. Class B receptors (i.e.

Angiotensin II receptor type 1A (AT1A) are characterized by a much stronger beta arrestin binding and thus much slower receptor recycling.^20,27

1.2.2.3 Beta arrestins and their new role as scaffolding proteins

In the past decade, a new role for beta arrestins as scaffolding proteins has been discovered. Besides their classical function as facilitators of receptor desensitization and internalization, they affect multiple signaling pathways upon binding to the GPCR. One of the first and most relevant discoveries of beta arrestin-mediated scaffolding function was the activation of MAPKs (Figure 3C). MAPKs describe a family of serine/ threonine kinases including ERK1/2, p38 kinases and c-Jun N-terminal kinases (JNK). These downstream effectors of MAPK are relevant for a huge variety of different cellular functions and signaling pathways.^27,32,33 For example, Defea et al. have shown that beta arrestins recruit the tyrosine kinase c-Src and that this recruitment is indeed necessary for the apoptosis- preventing effects of substance P.³⁴ Next, Lutrell et al. described the formation of a complex consisting of beta arrestin 2, ERK1/2, Raf-1 and MEK-1 upon activation of the AT1A receptor.³⁵ These pioneer discoveries gave rise to a completely new field of research about the effects of beta arrestins beyond desensitization and internalization. These findings indicate that beta arrestins can function as scaffold proteins for multiple signaling pathways by forming protein complexes and recruiting signaling molecules.²⁷

1.2.3 GPCR downstream signaling: G protein subtypes and their role in tone regulation of vessels While GRKs and beta arrestins represent the main regulators of GPCR function, signal transduction from receptor activation to actual cellular responses is mainly driven by G protein activation. The inactive G protein is a heterotrimeric complex consisting of a Gα and a Gβγ subunit. When both subunits are bound, the Gβγ subunit acts as a negative regulator for the Gα subunit. Upon G protein activation, both subunits dissociate and activate a wide array of signaling pathways. Gα subunit

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Introduction

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downstream targets include ACs, PLCβ and RhoA which modulate vascular tone in various ways.

Signaling of the Gβγ subunit appears similarly complex as Gα subunit signaling, but only little is known about its actual impact on tone regulation. The ability to recruit GRKs upon activation of the GPCR, thus participating in the modulation of the GPCR turn-off, appears to be a crucial mechanism of the Gβγ subunit.³⁶ Most GPCRs couple to different G proteins, which makes the understanding of signal transduction and downstream signaling targets very complex.³⁷ According to the topic of my thesis, the following chapters will introduce most relevant Gα subunit signaling pathways in vascular tone regulation.

1.2.3.1.1 Activation of Gαq/11 and Gα12/13 promotes contraction in VSMCs

Figure 4: Gαq/11 and Gα12/13 downstreamsignaling in VSMC

Activation of Gαq/11 and Gα12/13-coupled receptors causes contraction of VSMCs. Gαq/11 activates phospholipase C (PLC), an ezyme class responsible for the cleaveage of phospohatidylinositol-4,5-bisphosphate (PIP2) in diacylglycerol (DAG) and inositol triphosphate (IP3). IP3 bindes to inositol triphospohate receptors (InsP3R), releasing Ca²⁺ from the sarcoplasmic reticulum to the cytosol. Increased cytosolic Ca²⁺ levels and DAG activate proteinkinase C (PKC), an enzyme that opens L- type Ca²⁺ channels and enables a Ca²⁺ influx. Further, PKC phosphorylates protein phosphatase 1 regulatory subunit 14A (CPI-17) and subsequently inhibits MLCP. Increased intracellular Ca²⁺ interacts with calmodulin (CaM) and promotes MLCK activity. In total, activation of MLCK and inhibition of MLCP result in increased phosphorylation of MLC20 and thus increased contraction of VSMCs. Activation of Gα12/13 promotes activation of the GTP-binding protein RhoAvia the guanine exchange factor RhoGEF: activated RhoA stimulates the Rho kinase (ROCK) that inhibits MLCP and thus promotes VSMC contraction.

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Activation of Gαq/11 stimulates the enzyme phospholipase Cβ (PLCβ) which subsequently breaks phosphatidylinositol-4,5-bisphosphate (PIP2) down into the second messenger molecules diacylglycerol (DAG) and inositol triphosphate (IP3) (Figure 4). Cytosolic IP3 causes Ca²⁺-release by activation of Inositol triphosphate receptors (InsP3R) on the surface of the sarcoplasmic reticulum.

DAG, together with Ca²⁺, recruits and activates protein kinase C (PKC).^10,38 Activated PKC phosphorylates different effectors i.e. protein phosphatase 1 regulatory subunit 14A (CPI-17) or L-type Ca²⁺ channels. Phosphorylated CPI-17 inhibits MLCP, while phosphorylation of L-type Ca²⁺ channels result in further increase of intracellular Ca²⁺ levels. Increased intracellular levels of Ca²⁺, as well as reduced MLCP activity, promote the previously described (chapter 1.1.3.1) cross-bridge mechanism and result in contraction of VSMCs.^10,38 Well-known Gαq/11-coupled receptors in VSMCs causing vasoconstriction are the muscarinic acetylcholine (M1 and M3) receptors, the adrenaline and noradrenaline (α1) receptor, the angiotensin II (AT1) and endothelin-1 (ETA, B) receptor or the prostaglandin E2 (EP1) receptor.

Activated Gα12/13 subunits signal via the guanine-exchange factor RhoGEF and are known to activate the small GTP-binding protein Ras homolog family member A (RhoA). RhoA activates the Rho kinase (ROCK) and ROCK phosphorylates the regulatory myosin-binding subunit of MLCP. Phosphorylation and thus inactivation of MLCP increases cross-bridge activity and leads to a Ca²⁺-independent contraction. ^10,38,39 While activation of Gq/11 results in a transient but strong contraction, activation of G12/13 is responsible for a sustained and tonic contraction. Both signaling pathways appear to be closely connected and classical endogenously released vasoconstrictors such as endothelin-1 or angiotensin 2 are described to signal via Gαq/11 and Gα12/13 simultaneously, suggesting a synergistic effect of Gαq/11

and Gα12/13 for Ca²⁺-dependent and Ca²⁺-independent vasoconstriction.^10,38–40

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1.2.3.1.2 Activation of Gαs and inhibition of Gαi/o promotes relaxation in VSMCs

Figure 5 Gαs and Gαi/o downstreamsignaling in VSMC

Activation of Gαs-coupled receptors promotes VSMC relaxation, while activation of Gαi/o inhibits VSMC relaxation. While Gαs activates the adenylyl cyclase (AC) and increases cAMP levels, activation of Gαi/o inhibits AC and decreases cAMP levels.

CAMP activates proteinkinase A (PKA) but it is degraded by phosphodiesterases (PDE). PKA phosphorylates phospholamban (PLB) and activates MLCP. Unphosphorylated PLB inhibits sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) activity while phosphorylation of PLB enhances SERCA activity and results in a Ca²⁺ uptake from the cytosol to the sarcoplasmatic reticulum. Decreased intracellular Ca²⁺ results in lowered Ca²⁺- CaM interaction and decreased MLCK activity. In total, an increased MLCP and a decreased MLCK activity upon Gαs-activation results in decreased phosphorylation of MLC20 and thus promotes relaxation of VSMCs.

Upon activation, Gαs subunits activate ACs, a class of membrane-bound enzymes that catalyze the transformation of adenosine triphosphate (ATP) to the second messenger molecule cyclic adenosine monophosphate (cAMP) (Figure 5). In contrast, activated Gαi/o subunits inhibit AC and thus lower cAMP levels.¹³ CAMP is able to directly activate protein kinase A (PKA).³⁸ PKA promotes relaxation of VSMCs in at least two ways: it activates MLCP leading to dephosphorylation of MLC20 and a lowered myosin- actin interaction. Furthermore, it phosphorylates phospholamban (PLB), an important controller of the intracellular calcium balance. Unphosphorylated PLB inhibits sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) activity. SERCA is responsible for Ca²⁺ reuptake in the sarcoplasmic reticulum. A phosphorylation of PLB by PKA leads to increased SERCA activity and to decreased cytosolic Ca²⁺

levels.^41,42 Both mechanisms, MLCP activation and Ca²⁺ reuptake result in vasorelaxation. Well-known

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examples for Gαs-coupled receptors are the beta1- and beta2-adrenergic (β1 and β2) receptors, while the alpha2-adrenergic (α2) receptor is Gαi/o-coupled.

Cellular cAMP levels are controlled by Gαs and Gαi/o -coupled receptors modulating AC activity.

However, also phosphodiesterases (PDEs) control cyclic nucleotide levels directly by degradation of the phosphodiester bond of cyclic nucleotides such as cAMP or cGMP resulting in AMP or GMP molecules (overview in Figure 6).³⁸ 11 families of PDEs are currently described in literature. These molecules enable a precise regulation of local cyclic nucleotide levels depending on their expression and distribution throughout the body. PDE isozymes commonly exist as dimers and various isoforms are further subdivided into different PDE families.^43,44

Figure 6: Classification of the PDE family.

PDEs may be classified with respect to their cyclic nucleotide target: PDE4, 5 and 8 are described as cAMP-specific. PDE5, 6 and 9 are known as cGMP-specific. Dual specificity is given for PDE1, 2, 3, 10 and 11. Further differences exist in regard to enzyme stimulation: PDE1 is known to be stimulated by Ca²⁺-CaM, while PDE2 can be stimulated by cGMP and PDE3 is inhibited by cGMP.

While some PDE families are cAMP or cGMP-specific, most are dualspecific and able to degrade both cyclic nucleotides at different hydrolysis rates (overview in Figure 6). Besides targeting cAMP or cGMP to a different extent, differences also exist in the stimulation mechanism: While PDE1 is stimulated by the Ca²⁺-CaM complex, PDE2 is stimulated by allosteric cGMP binding and the cAMP degrading capacity of PDE3 is dampened by cGMP binding. These examples emphasize that PDEs -besides direct cyclic nucleotide degradation- further participate in the cAMP/cGMP crosstalk and thereby allow an interplay of different signaling pathways.⁴³

1.2.4 The canonical NO-cGMP-PKG signaling pathway in VSMCs

While cAMP and subsequent downstream signaling is directly modulated by Gαs and Gαi/o activation, the NO-cGMP-PKG signaling pathway is only indirectly modulated by GPCR activity. The NO-cGMP-PKG signaling pathway plays a critical role in VSMC tone regulation as NO is one of the strongest endogenous vasorelaxants. This signaling axis connects endothelial and vascular smooth muscle cells.⁴⁵

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Figure 7 Signaling pathway of NO-mediated VSMC relaxation

Endothelial NO production evokes relaxation of VSMCs. Various stimuli such as activation of Gq-coupled receptors or shear stress increase intraendothelial Ca²⁺ levels. Ca²⁺ binds to CaM and activates the endothelial NO synthase (eNOS). NO is generated by eNOS and difuses into the VSMC where it activates the soluble guanylyl cyclase (sGC). The sGC enzyme generates cGMP that can be degraded by PDE. PKG is activated by increased cGMP levels and affects multiple signaling pathways resulting in vasorelaxation. It opens K⁺ channels, causing a K⁺efflux-mediated hyperpolarization of the VSMC.

The hyperpolarization reduces L-Type Ca²⁺ channel-mediated Ca²⁺ influx. Additionally, it further phosphorylates and thus inhibits L-type Ca²⁺ channels directly, resulting in decreased Ca²⁺ influx. It phosphorylates PLB and InsP3R- associated PKG substrate isoform A (IRAG). Phosphorylation of PLB enables SERCA activity and results in Ca²⁺ uptake from the cytosol to the sarcoplasmatic reticulum. Phosphorylated IRAG prohibits Ca²⁺ release from the sarcoplasmic reticulum via InsP3R.

Decreased intracellular Ca²⁺ results in lowered Ca²⁺- CaM interaction and decreased MLCK activity. Increased MLCP and decreased MLCP activity result in relaxation of the VSMC.

1.2.4.1 Activity of eNOS controls NO production in the vascular endothelial cell

NO is a small gaseous molecule with a very short half-life that is generated by either neuronal, inducible or endothelial nitric oxide synthase (nNOS; iNOS; eNOS). ENOS is responsible for the NO production in the vascular system. The eNOS enzyme is modulated by intraendothelial Ca²⁺, Ca²⁺-CaM binds to a corresponding domain of the eNOS enzyme leading to NO generation. In endothelial cells intracellular Ca²⁺ is regulated by various different stimuli. For example, humoral agonists such as acetylcholine cause a Ca²⁺ release via activation of the Gαq-coupled endothelial M3 receptors while physical stimuli such as shear stress increase Ca²⁺ levels by a mechanism not yet understood completely.⁴⁶ Upon formation in the endothelial cell, NO diffuses into the smooth muscle cell and activates the sGC enzyme selectively.

1.2.4.2 The sGC as a unique and important regulator of VSMC relaxation

cGMP is generated in VSMCs by two different forms of guanylyl cyclases, the soluble guanylyl cyclase (sGC) and the particulate guanylyl cyclase (pGC). The pGC is a membrane-bound receptor and different

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isoforms exist in the human body. Known agonists are natriuretic peptides which can be further subcategorized into atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and natriuretic peptide type C (CNP).⁴⁷

The sGC is a cGMP-generating enzyme located in the cytoplasm of the cell. It is a heterodimeric protein consisting of an α- and a β- subunit. Acting as intracellular NO receptor, activation of the enzyme by its endogenous ligand NO causes a conformational change leading to the formation of cGMP.⁴⁸

Figure 8: Structure of the soluble guanylyl cyclase (sGC).

The sGC heterodimer consists of an α- and a β-subunit. Each subunit contains a C-terminally located catalytic domain, a central domain responsible for dimerization and a N-terminally located heme-nitric oxide binding domain (H-NOX). The β- subunit contains an N-terminally located heme group, which enables binding of the endogenous ligand NO. NO binding causes a conformational change of the enzyme, leading to increased catalytic activity at the c-terminus.

Two different α subunits are described. α1 is found to be highly expressed in all sGC-expressing cell types, while α2 is primarily expressed in the brain. Even though there is genetic evidence that a β2

subunit exists, the β1 subunit is the only physiologically relevant binding partner for the α subunits in the human body. Homodimers are existent, however, only heterodimers are catalytically active.⁴⁹ The heterodimer can be structurally divided into three parts: The catalytic C-terminus responsible for cGMP generation, the central part mainly involved in dimerization and the N-terminus as the NO- binding site.

The β subunit contains a heme-binding domain located at its N-terminal site that enables NO binding.

The prosthetic heme group is mandatory for sGC activation by endogenous NO and removal of the heme group abolishes any NO-mediated activity.^49,50 The prosthetic heme group at the β subunit contains a five-coordinated ferrous (Fe²⁺) heme with a histidine (His-105) as its axial ligand and coordinating residue. Binding of NO occurs at the sixth position of the complex and results in an intermediate NO- Fe²⁺-His complex. Next, a conversion to a five-coordinated nitrosyl heme complex is

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described and the histidine bond is broken, resulting in the conformational change of the enzyme and the increased catalytic activity.^49,51 The histidine (His-105) as the axial ligand as well as the iron ion in its ferrous state is necessary for proper sGC function: mutations in the histidine site or oxidation to the ferric form (Fe³⁺) lead to an insensitivity of the sGC enzyme for its endogenous ligand NO.^52,53

Besides the endogenous ligand NO that can be also produced by synthetic NO donors, pharmacological sGC activators and stimulators exist that reportedly activate sGC by different mechanisms.⁴⁹

1.2.4.3 Role of cGMP in vascular tone regulation

The cyclic nucleotide cGMP is degraded by PDEs. Thus, PDEs remain a crucial controller of cGMP- mediated vasorelaxation and cGMP-specific PDEs (i.e. PDE5) are established targets of modern pharmacotherapy.⁵⁴ Effects of cGMP on vascular tone are primarily mediated by activation of protein kinase G (PKG), while also cGMP-gated ion channels and allosteric PDE modulation should be mentioned. By binding to allosteric sites of the PKG enzyme, cGMP increases PKG phosphotransferase activity and affects various downstream targets. There exist two families of PKG, but only PKG1 is involved in NO-mediated cGMP signaling. There are two isoforms of PKG1 (PKG1α and PKG1β), both isoforms exist as homodimers. They are activated by allosteric binding of cGMP in the PKG regulatory domain, resulting in increased phosphotransferase activity.⁵⁵ Most relevant interactions for the relaxation of VSMCs will be introduced:

PKG is a modulator of PLB and MLCP, resulting in increased sarcoplasmic Ca²⁺reuptake and decreased actin-myosin interaction. Furthermore, PKG opens Ca²⁺ -sensitive K⁺ channels and the gradient- dependend K⁺ efflux causes hyperpolarization of the VSMC. This hyperpolarization decreases Ca²⁺

influx via L-Type Ca²⁺ channels and in addition PKG directly inhibits these Ca²⁺ channels by phosphorylation. Finally, PKG phosphorylates the InsP3R -associated PKG substrate isoform A (IRAG) at the sarcoplasmic reticulum, resulting in decreased Ca²⁺ release.⁵⁶ In total, PKG lowers intracellular Ca²⁺

and increases MLCP activity, resulting in relaxation of the VSMC (Figure 7).

Intracellular crosstalk is given by interaction with PKA, RhoA, sGC and PDE5 activity.⁵⁵ The interaction of PKG with PKA and RhoA represents a cross-talk between the cGMP signaling pathway and GPCR signaling. Inhibition of sGC activity and increased PDE5 activity represent a negative feedback mechanism for the sGC-cGMP-PKG signaling axis.⁵⁵

The paracrine effect of endothelial NO and the strong sGC expression in VSMCs reveal the NO-sGC- PKG signaling pathway as a relevant signaling axis for vasorelaxation. In contrast to the GPCR-mediated cAMP signaling pathway in VSMCs, generation of cGMP by NO is indirectly GPCR-dependent: while

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generation of NO by the eNOS enzyme in the endothelium is partially driven by GPCR (i. e. M3

receptors) activation, the NO signaling cascade does not rely on GPCR activation in the VSMC.

Disturbances in the NO-sGC-PKG signaling pathway are relevant in various cardiovascular diseases.

Therefore the sGC enzyme is a promising target for specific pharmacotherapy.⁴⁵ An indication for sCG modulators is pulmonary arterial hypertension (PAH), where NO-mediated vasorelaxation represents current state-of-the-art therapy. The current role of beta arrestins in PAH is unknown.

1.3 Pulmonary arterial hypertension (PAH): a paradigm of dysregulated (pulmonary) vascular physiology

1.3.1 PAH and its clinical appearance

PAH is a severe disease with an incidence ranging from 2.0 to 7.6 cases per million adults and year. It is characterized by a dynamic obstruction due to vasoconstriction of PAs and by structural obstruction due to adverse vascular remodeling.^57,58 PAH is classified as a sub form of pulmonary hypertension (PH). PH is defined as a hemodynamic state characterized by an increased (≥ 25 mm Hg) resting mean pulmonary arterial pressure (PAPm).

The different PH subforms substantially differ in terms of incidence, relevance and treatment.⁵⁹ The WHO classifies PH into 5 groups depending either on its origin or associated malfunctions.

Table 1: WHO-classification of PH WHO-classification of PH

WHO group 1 WHO group 2 WHO group 3 WHO group 4 WHO group 5

pulmonary arterial

hypertension (PAH) PH due to left heart

disease PH due to lung disease Chronic thromboembolic PH (CTEPH)

PH of uncertain multifactorial

mechanism

*content of Table 1 has been modified from Hoeper, M. et al. 2017 - Pulmonary Hypertension. ⁵⁹,⁶⁰

Cardinal symptoms of PH include increased exercise dyspnea, fatigue, edema and exercise-induced syncope.⁵⁹ PH is often caused by comorbidities such as left heart or lung disease.

1.3.2 PAH as a unique panvasculopathy in the PH setting

PAH is primarily caused by vasculopathies affecting the distal PAs.⁶¹ It is characterized by pathophysiological alterations of all layers of the vascular wall.⁶¹ Especially dysfunctional endothelial cells, smooth muscle cells, fibroblasts and immune cells contribute to disease progression.⁵⁷ An abnormal muscularization of distal, normally non-muscularized PAs, is an early sign for PAH development. In later stages, hyperplasia of the intima is described and this is linked to insufficient NO

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generation as well as formation of abnormal channels in the vessel lumen.⁶² A fully- developed PAH shows a strong phenotype of proliferation of endothelial cell in the intima, of smooth muscle cells in the media and of fibroblasts in the adventitia. This potentially leads to a complex interplay of endothelial dysfunction, vascular obstruction and fibrosis-induced vascular stiffening.⁵⁷

1.3.3 Treatment of PH

PH is characterized by a plethora of different clinical symptoms and therapy greatly differs between different groups of PH. For the treatment of PAH six classes of drug (Table 2) are currently approved.⁶³ Treatment of PH group 2 and 3 targets primarily underlying diseases, namely left heart and lung disease, while drugs approved for PAH (PH group 1) treatment seem to be ineffective for these types of PH.⁵⁹ Treatment of choice for chronic thromboembolic pulmonary hypertension (CTEPH) is surgical pulmonary endarterectomy.

Table 2: Drug classes for PAH treatment Drug classes for treatment of PAH Calcium channel

blockers

Endothelin receptor antagonists

Phosphodiesterase type 5(PDE5)

inhibitors

Guanylate cyclase stimulators

Prostacyclin

analogues IP receptor agonists

*content of Table 2 has been modified from Galiè, N. et al. 2015:

ESC/ERS Guidelines for the diagnosis and treatment of pulmonary hypertension.⁶³

To date PAH remains incurable. The PAH treatment aims at symptom control and prevention of disease progression.⁵⁹ The selection of specific drug classes (Table 2) for mono or combi treatment is highly dependent on the severity of the PAH as well as the individuals response to the treatment.⁵⁹

Besides the severity of diagnosed PAH also the clinical phenotype determines PAH treatment.

Moreover also the course of the disease (typical or nontypical) as well as the bioavailability of oral drugs compared to intravenously applied drugs determine the choice of medication.⁵⁹

1.3.4 Molecular mechanisms of drugs used in PAH treatment 1.3.4.1 Calcium channel blockers

Increasing Ca²⁺ concentrations in VSMCs lead to cross-bridge formation between myosin and actin and thus result in vasoconstriction.⁴² Inhibition of L-type Ca²⁺-channels by calcium channel blockers in the cell membrane results in lower intracellular Ca²⁺ concentrations and reduces vasoconstriction.⁶⁴ 1.3.4.2 Endothelin receptor antagonists

The polypeptide endothelin-1 is a very strong endogenous vasoconstrictor and secreted mainly from endothelial cells.⁶⁵ Endothelin-1 binds to different GPCRs, however vascular vasoconstriction is mainly driven by activation of Gq/11-coupled ETA receptors. Activation of this receptor in VSMC results in

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vasoconstriction by increasing Ca²⁺ concentrations released from the sarcoplasmic reticulum and by L- type Ca²⁺ -channel dependent influx.⁶⁶ Inhibition of the ETA receptor via endothelin receptor antagonists reduces vasoconstriction of VSMCs.

1.3.4.3 sGC stimulators and PDE inhibitors

Previously introduced endothelin receptor antagonists and Ca²⁺channel blockers reduce persisting vasoconstriction. An alternative therapeutic strategy is to enhance signaling pathways that mediate vasorelaxation, i.e. by the endothelium-dependent vasorelaxants NO and prostacyclin. As previously explained, endothelium-derived NO activates the sGC and increasing cGMP levels result in vasorelaxation. This sGC-mediated vasorelaxation can be pharmacologically induced by sGC activators.⁵⁴ Furthermore, cGMP degradation can be reduced by inhibition of PDE5.⁶⁷ Both drug classes - sGC activators and PDE5 inhibitors - are used for the treatment of PAH with the aim of increased vasorelaxation.

1.3.4.4 Prostacyclin analogues and IP receptor agonists

Prostacyclin is another important endogenous vasorelaxant secreted by the endothelium. Released prostacyclin activates Gs-coupled prostacyclin receptors (IP) on the surface of smooth muscle cells resulting in increasing cAMP levels causing vasorelaxation via activation of PKA.⁶⁸ Endogenous prostacyclin has only a short half-life. Therefore stable prostacyclin analogues and selective IP- receptor-agonists have been developed for and are currently used in the treatment of PAH.⁶³

Remarkably, most of today`s approved drugs for PAH treatment either target GPCRs (endothelin receptor antagonists, prostacyclin analogues, IP agonists) directly or affect second messenger modulation (calcium channel blockers, PDE inhibitors, sGC stimulators). Furthermore, the development of PAH appears to be closely related to a disbalance of GPCR expression.⁶⁹

Clinical trials classify PAH treatment with currently available drugs only as modest in regard to improvement of functional readouts (i.e. 6-minute walking test) and hemodynamics.^57,70 This underlines the urgent need for a better pathophysiological understanding of the disease and subsequent development of new pharmacological tools decreasing mortality.

1.4 Potential role of beta arrestins for vascular and lung physiology

Beta arrestins are well-known regulators of GPCR signaling and thus affect many physiological and pathophysiological processes that involve GPCR activation. The beta arrestin-mediated desensitization and internalization of GPCRs has been investigated and generation of genetically modified mice lacking beta Arr1 or 2 has allowed for intensive beta arrestin research in animal models. In contrast, the idea

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of beta arrestins acting as scaffolding molecules is rather new and knowledge in this research field is still expanding.⁷¹

1.4.1 The role of beta arrestins in the functionality of airways smooth muscle cells

Relaxation of airway smooth muscle cells (ASMC) is essential for the management of bronchospasm in obstructive lung diseases. Desensitization and thus loss of response of beta2-adrenergic receptor signaling due to excessive inhalation of beta2-agonists in the treatment of asthma is one of the best- known examples of receptor downregulation in modern pharmacology. Deshpande et al. have addressed this desensitization phenomenon in an extensive study. They were able to show that the ablation of the beta Arr2 gene increases beta2-agonist-mediated ASM relaxation in murine tracheal rings. Moreover they demonstrated that the classical desensitization of beta adrenergic receptors by beta arrestins is of functional relevance for murine bronchorelaxation in vivo.⁷² In a follow-up study, Pera et al. took a closer look at the different beta arrestin subtypes and their role for GPCR function in ASMC. They underline previous findings by proving that selective beta Arr2 knockdown or knockout (KO) augments beta2-adrenergic receptor signaling and function. Further, they highlight that selective beta Arr1 knockdown or KO does not affect beta2-adrenergic receptor signaling but selectively inhibited M3 muscarinic receptor signaling.⁷³ Expanding the view of beta arrestin function for asthma development beyond bronchorelaxation, Walker et al. claim that beta Arr2 is required for the development of allergic asthma. They were able to show that allergen-sensitized mice with genetic beta Arr2 deletion do not accumulate T lymphocytes in their airways and are protected from asthma- associated inflammation. This early regulation of the inflammatory cascade by beta Arr2 underlines the influence of beta arrestins on the development of allergic asthma and its potential as a future drug target.⁷⁴

All these findings elucidate the manifold roles of beta arrestins in asthma and ASMC function. While beta Arr2 is considered to play a crucial role for bronchorelaxation and the response for asthmatic bronchodilative therapy, not much is known yet about the role of beta Arr1 in this setting.

1.4.2 Beta arrestins in the context of PAH

Little is known about the role of beta Arr1 in PAH. Only very recently, Ma et al. published a study on the role of beta Arr1 in the crosstalk between GPCRs and the vascular endothelial growth factor receptor 3 (VEGFR3) in pulmonary vascular endothelial cells. Previously, impaired VEGFR3 signaling has been linked to PH, because endothelium-specific knockout of Vegfr3 was shown to exacerbate hypoxia-induced PH in mice.⁷⁵ Upon hypobaric hypoxia, mice lacking beta Arr1 developed a more severe PH compared to wildtype and beta Arr2-/- mice, suggesting a protective role of beta Arr1 for

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PH development. Ma et al. further suggest an interaction of both beta Arr1 and VEGFR3 as a rational cause for the development of PH.⁷⁶ They claim that a direct interaction between beta Arr1 and VEGFR3 promotes VEGFR3 signaling and decreases VEGFR3 internalization. When beta Arr1 is lacking in pulmonary arterial endothelial cells, VEGFR3 signaling is reduced. This observation serves Ma et al. as central explanation for the pathogenesis of PH.⁷⁶

This publication by Ma et al. is the first and only evidence so far for a distinctive role of beta Arr1 in the development of PH. While intensively focusing on the VEGFR signaling in the endothelium, it describes a strong phenotype of PH in beta arr1-/- mice. This underlines the role of beta arrestins as adaptor proteins by proving direct interaction with non-GPCR targets such as the VEGFR3.